Dopa decarboxylase (DDC; aromatic-L-amino-acid decarboxylase; aromatic-L-amino-acid carboxylase, EC 4.1.1.28) was purified from rat liver and its partial sequence was determined. Synthetic oligonucleotides were used to construct and screen rat liver cDNA libraries, and three clones were isolated and sequenced. The 2 kilobases of DDC cDNA cloned consisted of a 5'-noncoding segment of 78 nucleotides, a coding region of 1440 nucleotides, and a 3'-noncoding region of 438 nucleotides. The encoded protein of 480 amino acid residues had a molecular weight of 54,000. A special feature of the primary structure of rat DDC was a repeating structure consisting of 29 amino acid residues. A sequence of 58 amino acid residues, including this repeating structure of rat DDC, was found to show homologies with those of rat tyrosine hydroxylase, human dopamine beta-hydroxylase, and bovine phenylethanolamine N-methyltransferase, other mammalian enzymes that synthesize catecholamines. These results indicate that catecholamine biosynthetic enzymes are structurally related and suggest that their homologous domains are important for catechol-protein interactions.

Detailed information on innervation of the histaminergic system in the brain is essential to an understanding of the physiological roles of this system. In a previous immunocytochemical study with antihistidine decarboxylase (HDC) antibody, we detected extensive networks of histaminergic fibers in many areas of the rat brain (Watanabe et al., '84). In the present study, we improved the immunocytochemical procedure and examined the detailed distribution of histaminergic innervation in the rat brain with anti-HDC antibody. As reported previously, the highest concentrations of fibers were found in the hypothalamic nuclei and medial forebrain bundle. With the modified procedure, we detected more dense networks of HDC-immunoreactive (HDCI) fibers. Furthermore, we demonstrated for the first time the existence of HDCI fibers in other regions, namely, the thalamic nuclei, median eminence, fimbria of the hippocampus, habenular nuclei, superior colliculus, nucleus of the optic tract, parabrachial nuclei, mesencephalic trigeminal nucleus, superior, lateral, and spinal vestibular nuclei, posterior lobe of the hypophysis, and vascular organ of the lamina terminalis. We also found dense transverse fibers in the retrochiasmatic area and supraoptic decussation, which suggests bilateral innervation of the histaminergic system. These results indicate that innervation of the rat brain by the histaminergic system is more extensive than observed previously.

L-DOPA decarboxylase [DDC, aromatic-L-amino acid carboxyl-lyase, EC 4.1.1.28] was purified 800-fold from rat liver by several column chromatographic steps. The enzyme (specific activity, about 6 mumol/min X mg protein) had a molecular weight of 100,000 and gave a single band with a molecular weight of 50,000 on SDS-polyacrylamide gel electrophoresis. Its isoelectric point was pH 5.7. The absorption spectrum in the visible region of the purified DDC showed maxima at 330 and 420 nm. Polyclonal and monoclonal antibodies against DDC were produced by using this purified protein as an antigen. Polyclonal anti-DDC serum immunoprecipitated the DDC activities of rat, guinea-pig and rabbit livers (about 1, 10, and more than 100 microliter of antiserum, respectively, were required for 50% precipitation of 2 nmol/min of activity of these enzymes). The monoclonal antibody, named MA-1, belonged to the IgG1 subclass and immunoprecipitated the DDC activities of rat and guinea-pig livers to the same extent (about 0.5 micrograms of IgG was required to immunoprecipitate 2 nmol/min activity of each enzyme), but it did not affect the rabbit enzyme. The antibody MA-1 detected DDC molecules of both the purified enzyme and crude homogenate of rat liver blotted onto a nitrocellulose sheet. Immunohistochemically this antibody also stained specific neurons in the substantia nigra, raphe nucleus and locus coeruleus of rat brain.

The existence of L-histidine decarboxylase (HDC, EC 4.1.1.22)-like immunoreactive (HDC-I) cells in guinea pig retina was demonstrated using antiserum raised against HDC purified from fetal rat liver. The anti-HDC antiserum partially cross-reacted guinea pig L-DOPA decarboxylase (DDC, EC 4.1.1.28), so the histaminergic neurons were carefully identified. Comparison of HDC-I and DDC-like immunoreactive (DDC-I) cell types in adjacent sections revealed that HDC-I structures were found in some horizontal cells and amacrine cells, and double-staining procedures with anti-HDC antiserum and monoclonal anti-DDC antibody showed that HDC-I horizontal cells had no DDC-I structures, but all the HDC-I amacrine cells had DDC-I structures. From the results, some horizontal cells (with HDC-like immunoreactivities but without DDC-like immunoreactivities) were concluded to be histaminergic.

Both rat L-histidine decarboxylase (HDC) and guinea-pig L-DOPA decarboxylase (DDC) were shown immunohistochemically and immunochemically to react with anti-rat HDC antibody. No cross-reaction was observed in immunoprecipitation experiments, but both anti-rat HDC antibody and anti-rat DDC antibody immunostained neurons in the substantia nigra, raphe nucleus and locus coeruleus of guinea-pig brain. Moreover, on immunoblotting, anti-rat HDC antibody recognized not only rat HDC but also guinea-pig DDC, but not rat DDC. However, anti-rat DDC antibody showed no immunohistochemical or immunochemical cross-reactivity with rat HDC.